Isolation, selection and propagation of animal transgenic stem cells

ABSTRACT

Animal stem cells are obtained and maintained by culturing cells containing, in the genome, a selectable marker. Differential expression of the selectable marker enables preferential survival and/or division of the desired stem cells compared to the non-stem cells.

This is a continuation of application Ser. No. 11/084,154, filed Mar.21, 2005, which is a continuation of application Ser. No. 09/537,562,filed Mar. 30, 2002, now U.S. Pat. No. 6,878,542, which is acontinuation of application Ser. No. 08/535,141, filed Dec. 29, 1995,now U.S. Pat. No. 6,146,888, all of which are incorporated herein byreference.

This invention relates to methods of isolating and/or enriching and/orselectively propagating animal stem cells, genetically modified animalcells and animals for use in said method, transgenic animals providing asource of such cells and selectable marker constructs for producinggenetically modified cells and transgenic animals.

Stem cells are progenitor cells which have the capacity both toself-renew and to differentiate into mature somatic tissues.

Embryonic stem cells are the archetypal stem cell, being capable ofdifferentiating to form the whole gamut of cell types found in the adultanimal. Such stem cells are described as pluripotential as they arecapable of differentiating into many cell types. Other types of stemcells, for example bone marrow stem cells and epidermal stem cells,persist in the adult animal. These stem cells have a more restrictedcapacity for differentiation.

In general, when required for research purposes or for medical use, stemcells have to be isolated from tissue samples by various fractionationprocedures, but even after careful segregation of cell types, these stemcell preparations consist of mixed cell types and while enriched forstem cells, include high proportions of differentiated cells which arenot categorised as stem cells.

Furthermore, most stem cells cannot be grown readily in culture and whenattempts are made to culture stem cells, the cells being cultured (whichordinarily contain a mixed population of cell types) grow at differentrates and stem cells rapidly become overgrown by non-stem cell types. Anexception is that embryonic stem cells from two specific strains of mice(129 and Black 6) can be cultured in vitro. Thus established lines ofembryonic stem cells can be obtained by culturing early (3½ day)embryonic cells from murine strain 129 and Black 6, or hybrids thereof.

There has developed a pressing need to isolate and maintain in vitroembryonic stem cells from other murine strains and more especially fromother species including other laboratory animals (e.g. rats, rabbits andguinea pigs), domesticated animals (e.g. sheep, goats, horses, cattle,pigs, birds, fish, etc.) and primates. Similarly, numerous medicalapplications for other pluripotential cells such as haematopoietic stemcells also demand their isolation and culture in vitro.

However hitherto the problems associated with producing cultures of stemcells including the problem of producing cell populations of asatisfactorily low degree of heterogeneity and the problem of overgrowthin culture of non-pluripotent cells have not been solved. A particularproblem associated with the continuing presence of certaindifferentiated cell types is that these can cause elimination of stemcells from the culture by inducing their differentiation or programmedcell death.

We have now developed a technique by which the aforementioned problemscan be overcome.

According to one aspect of the invention there is provided a method ofisolating and/or enriching and/or selectively propagating animal stemcells, which comprises maintaining a source of said cells under cultureconditions conducive to cell survival, characterised in that the sourceof cells includes cells containing a selectable marker which is capableof differential expression in (a) stem cells and (b) cells other thanthe desired stem cells, whereby differential expression of saidselectable marker results in preferential isolation and/or survivaland/or division of the desired stem cells. In the context of thisinvention, the term “animal cell” is intended to embrace all animalcells, especially of mammalian species, including human cells.

Examples of stem cells include both unipotential and pluripotential stemcells, embryonic stem cells, gonadal stem cells, somatic stem/progenitorcells, haematopoietic stem cells, epidermal stem cells and neuronal stemcells.

In carrying out the method of the invention, the source of cells mayinclude pluripotential cells having a positive selectable marker andexpression of the said marker is used to permit isolation andmaintenance of the pluripotential cells. Alternatively, the source ofcells may include a negative selectable marker which is expressed incells other than the desired pluripotential cells and is usedselectively to deplete the source of cells of cells other than thedesired pluripotential cells.

The selectable marker may, for example, be a foreign gene, a cellulargene or an antibiotic resistance gene such as for example the bacterialneomycin resistance gene.

Alternatively the selectable marker may be a growth stimulating gene,for example an immortalising gene, an oncogene or a gene coding for thepolyoma or SV40 T antigens or derivatives thereof, or the selectablemarker may be a gene coding for a growth factor or a growth factorreceptor or a signal transducing molecule or a molecule that blocks celldeath.

In one particular embodiment the isolation and/or enrichment and/orselective propagation of the desired pluripotential cells is dependenton the presence of cells other than the desired pluripotential cells andthe simultaneous maintenance of both cell types is dependent onexpression of a selectable marker, in one or the other cell population,which is capable of rescuing cells that do not express the marker butwhich neighbour cells which do themselves express the marker. In thisinstance, the selectable marker may, for example, be the hypoxanthinephosphoribosyl tranferase (HPRT) gene.

In another embodiment the selectable marker may be a gene encoding aproduct which is toxic per se, or a toxic gene product which isconditionally active in combination with a suicide substrate. An exampleof such a gene product is a herpes simplex virus thymidine kinase(HSV-TK) in combination with ganciclovir.

Expression of the selectable marker may be achieved by operativelyinserting the selectable marker into an expression construct prior tointroduction to the cell source, in which case expression of theselectable marker can result from the introduction of either a stable ortransiently integrated construct. Alternatively, expression of theselectable marker results from operatively inserting the selectablemarker into an endogenous gene of the cell source.

Various means of introducing the selectable marker may be employed,including introduction into the cells by transfection, lipofection,injection, ballistic missile, viral vector or by electroporation.

The source of the cells may be a single cell such as a fertilizedoocyte, or it may comprise a mixture of cells, such as cells derivedfrom an embryo, blood or somatic tissue of a normally bred or transgenicanimal or cell line. In the latter case the selectable marker may beincorporated into the transgenic animal's genome.

Most preferably, in carrying out the method of the invention a gene orgene fragment operatively linked to and regulating expression of theselectable marker is/are associated with a pluripotential stage ofcellular development. Such a gene or gene fragment may be active inpluripotential cells of the developing embryo, especially in the innercell mass and/or primitive ectoderm, or may be active in adult stemcells.

In preparing a source of cells for use in accordance with the inventionone of the following protocols may advantageously be adopted:

-   -   introducing into a source of cells containing stem cells, a        selectable marker construct, wherein said selectable marker        construct is adapted to operatively link to an endogenous gene        which provides said differential expression, or    -   introducing into a source of cells containing stem cells, a        selectable marker construct, wherein said selectable marker        construct has been previously linked to one or more gene        fragments which provide said differential expression.

The genetic marker preferably comprises a selectable marker operativelylinked to a promoter which is differentially active in the desiredpluripotent cells (e.g. primitive ectoderm). By “selectable marker” ismeant a selectable gene which may be a foreign gene or a cellular genewhich is not naturally expressed, or such a gene which is naturallyexpressed, but at an inappropriate level, in the target cellpopulations. This gene in use acts as a selection marker by adapting thephenotype of the target cell population in such a way that cells withthe so-adapted phenotype may be enriched or depleted under particularculture conditions.

In the case where stem cells are embryonic cells it is preferred thatthe selectable marker is operatively linked to a promoter which isdifferentially active in stem (e.g. primitive ectoderm, primordial germcells) and non-stem cells. Promoter and other cis-regulatory elementsmay be included in the expression construct prior to introduction intothe cells or by targeting promoter-less constructs into specific genesby site specific recombination.

A wide variety of gene products may be relied upon for selectiveisolation and propagation of the desired stem cells, including markerswhich are designed to protect the desired cells from the effects of aninhibiting factor present in the culture medium. In this instance, theinhibiting factor can, for example, be an antibiotic substance whichinhibits growth or reproduction of cultured cells, not expressing thegene (i.e. cells other than the desired cells). The selectable marker(e.g. HPRT) may also provide protection both for the desired cells inwhich it is expressed as well as other closely associated cells by meansof metabolic rescue.

Alternatively the selectable marker may selectively permit the growth ofstem cells. In this instance the marker may encode a growth factor, agrowth factor receptor, a transcription factor, an immortalising or anoncogenic product (e.g. temperature sensitive simian virus 40 Tantigen).

Alternatively, the selectable marker may be a cell surface antigen orother gene product which allows purification or depletion of expressingcells for example by panning or fluorescence-activated cell sorting(FACS). The invention thus enables stem cell populations to beobtained/maintained having a satisfactory degree of homology.

Alternatively the selectable marker may be a conditionally toxic genefor instance herpes simplex virus thymidine kinase [HSV-TK]. In thisinstance expression of the selectable marker is directed to cells otherthan the desired cells and not to stem cells. Cells other than thedesired phenotype may be selectively depleted by addition of a lethalsubstrate (e.g. ganciclovir).

The genetic marker may be introduced into the source of cells by avariety of means, including injection, transfection, lipofection,electroporation or by infection with a viral vector.

Further, the source of cells may be produced by transfectionextemporaneously, or the source of cells may be derived from atransgenic animal, e.g., the founder transgenic animal or an animal atleast one ancestor of which has had the aforementioned genetic markerintroduced into its genetic complement. In such transgenic animals it ispossible for the marker to pass down the germ line and eventuallyresults in the production of progeny, from the tissues of which(especially from the embryonic tissue) the required source of cells canbe derived.

Thus according to further aspects of the invention, there is provided ananimal cell capable of being cultured under appropriate selectiveculture conditions so as to enable isolation and/or enrichment and/orselective propagation of stem cells, characterised in that said cellcontains a selectable marker wherein differential expression of theselectable marker in (a) the desired stem cells and (b) cells other thanthe desired stem cells enables selective growth of the desired stem cellto occur.

The invention further provides an animal cell capable of being culturedunder selective culture conditions so as to grow as stem cells,characterised in that said cells contain stem cells containing a geneticmarker, whereby a gene product associated with the genetic marker isproduced and which under said culture conditions causes selectivesurvival and/or division of the desired stem cells to occur.

The animal cells according to this aspect of invention are preferablycharacterised by possessing the preferred characteristics describedabove.

The invention further provides according to another aspect thereof, atransgenic animal having genetic characteristics such that it or itsprogeny, during embryonic development or later life, constitute a sourceof animal pluripotential cells as defined above.

Such transgenic animal may be produced according to the invention byintroducing a genetic marker into a fertilised oocyte or an embryoniccell, or an embryonic stem cell in vitro the genetic marker having thecharacteristics defined above, and utilising the resulting transformedoocyte or embryonic cell as a progenitor cell for the desired transgenicanimal.

Vectors for use in producing an animal cell defined above form a furtheraspect of the invention.

Thus the invention further provides vectors for use in geneticallymodifying animal cells so as to produce transformed cells suitable foruse as the source of cells for the method referred to above, said vectorcomprising a first genetic component corresponding to said selectablemarker and a second genetic component which in the genetically modifiedanimal cells (1) results in the said differential expression of theselectable marker from either a transiently or stably integratedconstruct or (2) enables site-directed integration of the selectablemarker into a specific gene so as to provide operative coupling of theselectable marker with targeted endogenous gene regulatory elements.

Such vectors may be in the form of expression vectors in which saidsecond genetic component includes control sequences which aredifferentially activated (a) in stem cells and (b) in cells other thanthe desired stem cells.

The invention covers vectors which when used to transform animal cellsbecome integrated into the animal genome as well as vectors which do notbecome so integrated.

The expression vectors referred to above may comprise a DNA sequencecoding for the afore-mentioned selectable marker operatively linked to agenetic control element, or sequence enabling targeting of apromoterless marker to an endogenous gene which is expresseddifferentially in the said stem cells and in cells other than thedesired stem cells.

For the generation of pluripotential embryonic stem cells the expressionconstructs preferably comprise a DNA sequence coding for said selectablemarker operatively linked or targeting to a genetic control element(s)which is associated with a stage of embryonic development prior todifferentiation of pluripotential embryonic cells. Most preferably thegenetic control elements derive from a gene specifically active in theinner cell mass of the mouse blastocyst, in primitive ectoderm, and inprimordial germ cells of the early embryo.

In more detail, the present invention has resulted in the development ofexpression constructs which direct specific expression of selectablemarkers in stem cells and not in differentiated cell types. Havingintroduced an expression construct by transfection or via the generationof transgenic animals, stem cells present within mixed cell populationscan be isolated by culturing in the presence of the selection agent invitro, or by otherwise manipulating the culture conditions.

One example of a gene which displays a suitably restricted stem cellexpression pattern and therefore may provide suitable “stem cellspecific” regulatory elements for the expression of a selectable markerin accordance with the invention is the Oct4 gene.

Octamer binding transcription factor 4 is a member of the POU family oftranscription factors (reviewed by Schöler, 1991). Oct4 transcription isactivated between the 4- and 8-cell stage in the developing mouse embryoand it is highly expressed in the expanding blastocyst and then in thepluripotent cells of the egg cylinder. Transcription is down-regulatedas the primitive ectoderm differentiates to form mesoderm (Schöler etal., 1990) and by 8.5 d.p.c. (days post coitum) is restricted tomigrating primordial germ cells. High level Oct4 gene expression is alsoobserved in pluripotent embryo carcinoma and embryonic stem cell lines,and is down-regulated when these cells are induced to differentiate(Schöler et al., 1989; Okamoto et al., 1990).

Selectable marker genes under the control of the Oct4 promoter may,according to the invention, be applied to the isolation of embryonicstem cell lineages. Furthermore, reports describing low level Oct4expression in some adult tissues (Takeda et al., 1992) may extend theutility of these expression constructs beyond embryonic stem cells toinclude other stem cells essential to tissue homeostasis and repair inother systems including the haematopoietic system. In the event thatOct4 is not expressed in somatic stem cells, other transcriptionalregulatory elements, such as those associated with the haematopoieticstem cell specific antigen CD34, may be utilised in a similar manner.

Two specific approaches are provided according to the invention forgenerating the desired spatial and temporal restrictions in transgenicexpression. The first approach is through the generation of transgenicanimals in which a partially characterised Oct4 gene promoter fragment(Okazawa et al., 1991) is employed to drive stem cell specifictranscription of the selectable marker. An appropriate selectable markeris the neomycin phosphotransferase gene which confers resistance to theantibiotic G418. An alternative is to utilise a selectable marker whichis associated with the production of a gene product which can counteracta deficiency in a metabolite, e.g. the hypoxanthine-guaninephosphoribosyl transferase (HPRT) gene in HPRT-deficient cells (Hooperet al., 1987). This approach may be advantageous in situations wherestem cells require continuous support from closely associateddifferentiated cells. In this instance direct cell contact will permitmetabolic rescue of the neighbouring support cells by the stem cellsdespite the lack of selectable marker gene expression in the supportcells.

The second approach utilises the endogenous Oct4 gene locus, andtherefore the associated Oct4 gene regulatory elements, to linkresistance marker gene expression as closely as possible with theendogenous Oct4 gene expression profile. This may be accomplished byhigh efficiency gene trap targeted mutagenesis of the Oct4 gene inembryonic stem cells. This approach provides more tightly regulatedcontrol of selectable marker gene expression by avoiding randomintegration site effects which often result in unpredictable expressionpatterns of randomly integrated constructs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of plasmid Oct-4-Neo-βS.

FIG. 2 illustrates the structure of plasmid Oct-4-Neo-βfos.

FIG. 3 illustrates the structure of the plasmid Oct-4-tgtvec.

EXAMPLE 1

1. Isolation of OCT4 Promoter Sequences:

We screened a strain 129 mouse genomic lambda library with a 330 bp5′Oct4 cDNA fragment. Several clones were isolated and screened byrestriction analysis and Southern blot hybridization. A 1.4 kb BamHI-Hind III fragment containing the Oct4 promoter element (Okazawa etal., 1991) was isolated from clone 1 and ligated into pBluescript IIKS(−) (Stratagene) to generate pOct4 (5′ genomic).

2. Construction of Plasmids:

To generate the Oct4-Neo promoter constructs an engineered Neomycinresistance gene (neo), designed to provide an Nco I restriction site atthe translation initiation codon, was isolated from pLZIN (Ghattas etal., 1991) as a 1.1 kb Xba I-Sph I fragment encompassingencephalomyocarditus virus internal ribosome entry site sequence(EMCV-IRES, Ghattas et al., 1991) and 5′-Neo coding sequences and clonedinto pSP72 (Promega Biotech). The Kpn I-Nco I EMCV-IRES sequence wasreplaced with a 1.3 kb Oct4 promoter fragment isolated from pOct4(5′genomic) by Kpn I and subsequent partial Nco I restriction digest.Neo3′-coding, rabbit β-globin gene (intron) and SV40 polyadenylationsequences were isolated as a 1.7 kb Sph I fragment from 6P-IRESNeo-βSand ligated into the Sph I site to generate Oct4-Neo-βS (FIG. 1). Togenerate the Oct4-Neo-fos construct (FIG. 2), an Oct4-Neo-βS Bam HIfragment incorporating the Oct4 promoter, neo gene and the rabbitβ-globin intron was inserted 5′ to a human c-fos genomic sequence. This1.7 kb genomic sequence (Bal I-Sph 1) encodes human c-fos mRNA 3′ codingand no-coding sequences previously associated with mRNA destabilization(Wilson and Triesman, 1988), and, the c-fos polyadenylation sequence.

The Oct4-neo construct (Oct4-tgtvec) is designed for targettedintegration into the Oct4 gene (FIG. 3). The Oct4 targetting constructcontains 1.7kb of 5′Oct4 gene sequence and 4.2 kb of 3′Oct4 genesequence. Following homologous recombination this construct incorporatesa IacZ-neomycin fusion gene (βgeo, encoding a bifunctional protein,Freidrich and Soriano, 1991) into the first intron of the Oct4 gene.Splicing from the splice donor sequence of the first exon-intronboundary to the integrated IRES-βgeo sequence is facilitated by theinclusion of a murine engrailed-2 splice acceptor sequence (Skarnes etal., 1992) immediately 5′ to the IRES-βgeo sequence. Translation of theβgeo cistron of the Oct4-βgeo fusion transcript is facilitated by theinclusion of the EMCV-IRES immediately 5′ to the βgeo coding sequence.

3. ES Cell Transfection and Colony Selection:

Mouse 129 ES cells (line CGR-8) were prepared and maintained in thepresence of Differentiation Inhibiting Activity (DIA) or LeukemiaInhibitory Factor (LIF) as described by Smith (1991). Plasmid DNA fortransfection was linearised by Sal I digest, ethanol precipitated andresuspended at 10-14 mg/ml in PBS. Following 10 hours culture in freshmedium, near confluent ES cells were dispersed by trypsinisation, washedsequentially in culture medium and PBS, and resuspended at 1.4×10⁸/ml inPBS for immediate transfection. Routinely, 0.7 ml of cell suspension wasmixed with 0.1 ml DNA containing solution and electroporated at 0.8 kVand 3.0 μFD using a Biorad Gene Pulser and 0.4 cm cuvettes.Transfections were plated on gelatinised tissue culture dishes at5-8×10⁴/cm² in growth medium for 16 hours prior to the addition ofselection medium containing 200 μg/ml (active) G418 (Sigma). Singlecolonies were picked 8-10 days post-transfection and transferred induplicate into 24 well tissue culture plates for further expansion ingrowth medium containing 200 μg/ml G418.

Clonal cell lines bearing the Oct4-Neo-βS and Oct4-Neo-fos constructs(referred to as Oct4-Neo cell lines) were grown for two days, washedtwice with PBS and the medium replaced with fresh G418 medium with orwithout DIA. Cell lines which grew normally in the presence of DIA butdid not survive in the absence of DIA were selected for expansion andfurther analysis.

Clonal cell lines bearing the Oct4-tgtvec targetting construct (referredto as Oct4-targetted cell lines) were expanded in duplicated 24 wellplates. Once confluent, one series of cells were frozen for storagewhile the reminder were analysed by Southern analysis.

4. Further Characterisation of Oct4-Neo and Oct4-Targetted Cell Lines:

Selected Oct4-Neo cell lines were assayed for ES cell growth anddifferentiation in DIA supplemented or non-supplemented medium atvarious G418 concentrations. Cells were plated at 1×10⁴/cm² in 12 welltissue culture plates in the various media preparations and cultured for6 days. Fresh medium was applied every 2 days until day 6 when cellswere fixed and stained as previously described (Smith, 1991.)Oct4-targetted cell lines positive by genomic Southern analysis wereanalysed by lacZ staining and growth and differentiation in DIAsupplemented or non-supplemented medium in 2OOpg/ml G418.

5. Production of Embryoid Bodies from Oct4-Neo Cell Lines:

Embryoid bodies were generated by the hanging drop method (Hole andSmith, in press) and maintained in suspension culture for 2, 4, 6 or 8days in the presence or absence of G418. Control embryoid bodies weregenerated from the parental cell line CGR-8 in the absence of G418.Embryoid bodies were then collected and transferred to gelatinisedtissue culture dishes to enable adherence and expansion of theaggregates for analysis of contributing cell types. All embryoid bodieswere maintained for 4 days in the absence of DIA and G418 prior toinspection.

6. Production of Chimeras from Oct4-Neo and Oct-4 Targetted Cell Lines:

Selected Oct4-Neo cell lines were cultured in the absence of G418 for 7days prior to embryo injection as previously described (Nichols et al.,1990). Briefly, blastocysts for injection were collected 4 d.p.c. fromC57BL/6 donors, injected with 10-20 cells and allowed to re-expand inculture prior to transfer to the uteri of pseudopregnant recipients.Chimeras were identified by the presence of patches of sandy coat colouron the C57BL/6 background. Male chimeras were test bred for transmissionof the Oct4-Neo transgene. Transgenic mice were then crossed ontodifferent genetic backgrounds.

7. Results

The Oct4-Neo-βS construct generated approximately 50 colonies/10⁶ cellstransfected while the Oct4-Neo-βfos construct generated approximately 10fold fewer colonies. Three clones were selected on the basis of theirdifferential survival in medium containing G418 (200 μg/ml) in thepresence or absence of DIA. All three cell lines displayed apparentlynormal growth rates in DIA-supplemented G418 containing media and diedwhen cultured in the absence of DIA in G418 medium. Cultures maintainedin DIA supplemented G418 medium grew as essentially pure ES cells whilecultures maintained in DIA supplemented medium in the absence of G418grew as mixed cultures of ES cells and differentiated progeny closelyresembling those of the parental CGR-8 line. Thus G418 selectioneliminates differentiated cell types and allows propagation of pure stemcell populations. The three cell lines selected were designatedOct4-Neo-βS18, Oct4-Neo-βS21 and Oct4-Neo-βfos11. These cell lines havebeen introduced into host blastocysts and resulting chimaeras may betest bred. Similar results were obtained with ES clones targetted withthe Oct4-tgtvec construct. In addition, histochemical staining of thesecultures for β-galactosidase activity confirmed that expression of βgeowas restricted to undifferentiated stem cells (Mountford et al, 1994).

Embryoid bodies were generated from the Oct4-Neo cell lineOct4-Neo-βpfos11 to examine the effect of G418 selection on mixed cellaggregates and to test the utility of the selection system in isolatingES cells from mixed cell populations. Embryoid bodies generated withboth the experimental cell line (Oct4-Neo-βfos11) and the parental cellline (CGR-8) and cultured in the absence of G418 were composed almostentirely of differentiated cells with few if any ES like cells. Incontrast, visual analysis of the expanded colonies revealed that theOct4-Neo-βfos11 embryoid bodies cultured in the presence of G418contained high proportions of ES cells. The feasibility of isolatingstem cells from differentiating systems is thus confirmed.

8. Summary

ES cells capable of germ line transmission have previously beenestablished from only 2 inbred strains of mice, 129 and C57BL/6.Combining the Oct4-neomycin selection scheme with established of ES cellisolation and propagation procedures (Evans and Kaufman, 1981; Martin,1981; Nichols et al., 1990; Yoshida et al, 1994) should enable ES cellline derivation from previously non-productive mouse strains and othermammalian species in which Oct4 is differentially expressed.

Selection against non-stem cell phenotypes in mixed cell populations maybe advantageous for several reasons. Firstly, selection againstdifferentiated cells in mixed populations provides a method forextensive stem cell enrichment. Secondly, selective removal ofdifferentiated cells prevents their overgrowth in the cultures. Thirdly,elimination of differentiated cells may enhance stem cell self-renewaldue to the loss of differentiation inducing activity associated withdifferentiated cells.

EXAMPLE 2 Rescue And Recovery of Pluripotential Stem Cells From Es CellEmbryoid Bodies

Methods

1. Cell Culture

ES cells were routinely maintained in medium supplemented withDifferentiation Inhibiting Activity (DIA) as described by Smith (1991).Embryoid bodies were formed by aggregation of ES cells in the absence ofDIA. The aggregates were produced by plating dissociated ES cells in 10μul or 30 μl drops of medium at a density of 100 cells/drop. Arrays ofdrops were plated on the lid of a 10 cm tissue culture dishes using amultipipettor. This was then inverted over the base of the dish, whichcontained 10 ml of water in order to maintain humidity, and the hangingdrops were cultured at 37° C. in a 7% CO₂ atmosphere.

2. Histology and β-Galactosidase Staining

Embryoid bodies were fixed in Bouin's solution and embedded in agar.Paraffin sections were then prepared by standard procedures and stainedwith haematoxylin and eosin. Alkaline phophatase staining of embryoidbody outgrowths was carried out using Sigma Kit 86-R. Histochemicalstaining for β-galactosidase was performed with Xgal as described(Beddington et al, 1986).

Results

3. Cell Lines and Selection System

Fos11 is a derivative of the ES cell line CGR8 which has beentransfected with the Oct4neofos construct. Fos11 cells express lowlevels of G418 resistance under control of the Oct4 proximal promoterelement, but differentiated progeny show no expression of the transgeneand are therefore sensitive to G418. OKO160 and OKO8 are derivatives ofthe ES-cell lines CGR8 and E14TG2a respectively in which an IRES-βgeopAcassette has been introduced into one allele of the Oct4 gene byhomologous recombination as described. OKO cell lines express highlevels of βgeo in the undifferentiated state and therefore stainstrongly with Xgal and are G418-resistant. Differentiated progeny loseexpression of βgeo and become negative for Xgal staining and sensitiveto G418. In monolayer cultures, Fos11 and OKO cells are maintained aspure ES cell populations by culture in the presence of DIA and selectionin G418. Under conditions which favour differentiation, however, such aslow density and absence of DIA (Smith, 1991), G418 selection results inthe complete elimination of these cultures over 3-5 days. Rb4O cells area derivative of CGR8 which are constitutively resistant to G418 due toexpression of neoR directed by the human β-actin promoter.

4. Formation of Embryoid Bodies in the Presence and Absence of Selectionagainst Differentiated Cells

Production of embryoid bodies by the conventional procedure (Doetschmanet al, 1986) of detachment of clumps of cells followed by aggregation inbulk suspension culture results in a mixed population of aggregates,heterogeneous in both size and differentiation status. In order toobtain more uniform and consistent development, embryoid bodies in thepresent study were formed by aggregation of defined numbers of cells inindividual drops of culture medium (see Methods). After 48 hours inhanging drop culture, the aggregates were transferred en masse intosuspension culture in the presence or absence of G418.

Under G418 selection against differentiated progeny aggregates stillformed from both Fos11 cells and the OKO clones. Although some deadcells appeared around the periphery of the aggregates, the bodiesthemselves increased in size during the culture period. Samples wereharvested periodically from the bulk cultures and processed forhistological examination. After several days embryoid bodies formed inthe absence of selection were mostly cystic and contained a variety ofmorphologically differentiated cell types. Undifferentiated cells wererarely apparent. By contrast, aggregates maintained under selectionshowed no indications of cellular specialisation and the bodies appearedto consist of solid balls of undifferentiated cells. The great majorityof cells in these undifferentiated aggregates appeared healthy andviable and there was no evidence of necrosis, although occasionalpyknotic nuclei, suggestive of apoptosis, were seen. Embryoid bodiesformed in G418 were noticeably smaller than their counterparts formed inthe absence of selection, however. This can be attributed to acombination of the lack of cyst development and the removal ofdifferentiated cells.

5. Persistence of Pluripotential Stem Cells in Embryoid Bodies FormedUnder Selection against Differentiated Cells.

The absence of any undifferentiated aggregates in control culturesimplied that it was unlikely that the effect of G418 was due toselection of a subpopulation of non-differentiating aggregates. In orderto exclude definitively this possibility, however, and also tofacilitate quantitative determination of the effects of G418 selection,a modified protocol was used which allows assessment of the behaviour ofindividual aggregates. Cultures were initiated in 30μl hanging drops inthe presence or absence of G418 and maintained in drop culture for 7-8days. Embryoid bodies were then transferred individually togelatin-coated 96-well tissue culture plates and the media diluted6-fold with media lacking G418. The stem cell maintenance factor DIA wasadded at this stage to allow expansion of any undifferentiated ES cellswhich were present. The cultures were allowed to attach and outgrow for48 hours then fixed and stained for alkaline phosphatase or forp-galactosidase as appropriate.

The data summarized in Table 1 show that in the absence of any selectionundifferentiated stem cells are almost completely eliminated fromembryoid bodies within 7 days of suspension culture. Outgrowthscontained a variety of morphologically differentiated cell types, butareas of cells with ES cell morphology were not observed. In the OKOcells expression of p-galactosidase is coupled to the stem cell-specifictranscription factor Oct4 (Mountford et al, 1994) and therefore servesas a marker of undifferentiated cells. Isolated Xgal-staining cells wereoccasionally seen in OKO outgrowths, but clusters of staining cells werenot detected under these conditions (but see Discussion).

The efficiency of embryoid body formation in G418 was identical to thatin non-selected cultures, essentially 100%. In marked contrast to theuntreated embryoid bodies, however, embryoid bodies established undercontinuous G418 selection gave rise to outgrowths consisting largely ofES cells. The undifferentiated nature of these cells was indicated bythe characteristic morphology of ES cell colonies and by staining withalkaline phosphatase and was confirmed by Xgal staining of the OKOoutgrowths.

Several outgrowths from embryoid bodies formed under selection werepicked and transferred to 2 cm wells. All of the colonies picked werereadily expanded into mass cultures of undifferentiated cells. Thesecultures remained dependent on DIA and differentiated in similar fashionto parental ES cells when plated in non-supplemented media. Furthermore,these derivatives differentiated efficiently into multiple cell types onaggregation, confirming their pluripotency.

These findings demonstrate that the selective elimination ofdifferentiated progeny results in the persistence of pluripotential stemcells in ES cell aggregates.

6. Stem Cell Extinction in Mixed Aggregates

The implication that differentiated progeny may be directly responsiblefor stem cell extinction in embryoid bodies was addressed further. Thebehaviour of OKO cells was assessed following formation of mixedaggregates with Rb40 ES cells which can differentiate in the presence ofG418. Rb4O cells express neomycin phosphotransferase constitutively andG418 selection has no discernible effect on their differentiation,either in monolayer culture or in aggregates. Hanging drop cultures wereestablished using a 3:1 ratio of OKO cells to Rb40 cells. Paraffinsections of mixed embryoid bodies revealed that they underwent extensivedifferentiation in both the absence and the presence of G418. Theeffective elimination of undifferentiated stem cells under bothconditions was confirmed by Xgal-staining of outgrowths (Table 1).

This result provides direct evidence that the presence of differentiatedprogeny induces the elimination of pluripotential stem cells. Thisimplies that certain differentiated stem cell progeny are a source ofinductive signals which either instruct further differentiation ofremaining stem cells or possibly induce them to enter apoptosis.

CONCLUSION

Aggregation induces ES cells to develop into differentiated structuresknown as embryoid bodies. Pluripotential stem cells rapidly becomeextinct in these embryoid bodies due to the efficient induction ofdifferentiation and possibly also to selective cell death. However, ifdifferentiated progeny are specifically eliminated from the aggregatesusing methods according to the invention, the stem cells persist and canbe propagated.

The findings detailed above constitute a clear demonstration thatthrough the use of a stem cell-specific selection system according tothe invention it is possible to recover stem cells from conditions whichwould normally force their elimination by either differentiation ordeath. TABLE 1 Disappearance of Persistence of Oct-4 Expressing ES Cellsin Embryoid Bodies. No. No. Xgal % +ve Culture G418* No Drops Outgrowths+ve Drops OKO8 − 25 25 0 0 OKO8 + 25 24 24 96 OKO160 − 30 30 0 0OKO160 + 30 30 30 100 OKO160:Rb40 − 30 29 0 0 OKO160:Rb40 + 30 30 0 0*500 μg/ml

EXAMPLE 3 Procedures for Establishing Embryonic Stem Cell Cultures fromMouse Embryos

Lines of transgenic mice were established in which the neomycinphosphotransferase gene conferring resistance to G418 is expressed withthe specificty of the Oct4 gene. The βS21 line harbour the Oct4neoμStransgene whilst in the OKO line the neo gene has been incorporated intothe endogenous Oct gene via gene targeting with the Oct4-tgtvecconstruct. These mice were outcrossed for two generations with MF1outbred albino mice and with inbred CBA mice. Neither of these mousestrains produce ES cells using standard procedures.

Four preferred procedures for isolating stem cells are described. In allcases the embryos are cultured in standard ES cell culture mediumsupplemented with either Differentiation Inhibiting Activity (Smith,1991) or interleukin-6 plus soluble interleukin-6 receptor (Yoshida etal, 1994). G418 is added at concentrations of 200 μg/ml-1 mg/ml.

Procedure 1

Blastocysts are flushed on the fourth day of pregnancy. Groups of 4-10blastocysts are cultured in 1 cm tissue culture wells under G418selection. Outgrowths are individually detached and dissociated withtrypsin as described (Nichols et al, 1990) after 4-6 days in culture andreplated in single wells. G418 selection is maintained. Colonies withthe characteristic morphology of ES cells which appear in the culturesover the next 14 days are picked and expanded under continuousselection.

Procedure 2

As Procedure 1, except that blastocysts are put into implantation delaybefore harvesting by ovariectomy of the dams on the third day ofpregnancy. Blastocysts are flushed 4 days after the ovariectomy.

Procedure 3

Post-implantation embryos between 5.5 and 7.5 days post-coitum areisolated and the primitive ectoderm separated by microdissection and/orprotease digestion. The primitive ectoderm is gently dissociated intoclumps of 20-50 cells which are then cultured as in Procedure 1.

Procedure 4

Embryos prepared as for Procedures 1, 2 or 3 are cultured in hangingdrops under G418 selection for a period of 5-7 days before transfer totissue culture wells and subsequent manipulation as in Procedure 1.

1-52. (canceled)
 53. A method of preparing a transgenic non-human animalcomprising: a) obtaining a desired stem cell by: i) culturing mammaliancells containing a nucleic acid construct including a selectable markeroperably linked to a promoter that preferentially expresses theselectable marker in mammalian stem cells; and ii) isolating mammalianstem cells expressing the selectable marker; and b) generating thetransgenic non-human animal therefrom.
 54. A method of preparing atransgenic non-human animal, said animal comprising a selectable markeroperatively linked to a promoter that preferentially expresses theselectable marker in mammalian stem cells, the method comprising: a)providing a blastocyst; b) providing an animal cell comprising at leastone nucleic acid construct encoding a selectable marker operably linkedto a promoter which preferentially expresses said marker in a mammalianstem cell; c) introducing the animal cell into the blastocyst; d)transferring the blastocyst to a recipient; and e) allowing an embryo todevelop to a chimeric animal to enable germline transmission of theselectable marker.
 55. The method of claim 54, wherein the mammalianstem cells are pluripotential stem cells or unipotential stem cells. 56.The method of claim 54, wherein the mammalian stem cells are embryonicstem cells, gonadal stem cells, or somatic stem cells.
 57. The method ofclaim 54, wherein the mammalian stem cells are murine stem cells. 58.The method of claim 54, wherein the nucleic acid construct is stablyintegrated or episomally maintained.
 59. The method of claim 54, whereinthe promoter is an Oct4 promoter.
 60. The method of claim 54, whereinthe promoter is a CD34 promoter.
 61. The method of claim 54, wherein theselectable marker is a gene product that allows purification of saidmammalian stem cells by fluorescent-activated cell sorting (FACS). 62.The method of claim 54, wherein the selectable marker is a fluorescentmarker.
 63. The method of claim 54, wherein the selectable marker is acell surface antigen.
 64. The method of claim 54, wherein the selectablemarker is a growth factor receptor.
 65. The method of claim 54, whereinthe selectable marker is an antibiotic resistance gene.
 66. The methodof claim 65, wherein the antibiotic resistance gene is a neomycinphosphotransferase gene.
 67. The method of claim 54, wherein the animalcells are embryonic stem cells.
 68. The method of claim 54, wherein theselectable marker is transmitted via the germline to progeny of saidtransgenic non-human animal.
 69. A method of isolating mammalian stemcells from a population of mammalian cells, comprising: a) obtainingcells from a transgenic animal prepared using the method of claim 53 orclaim 54, wherein the mammalian cells contain a nucleic acid constructincluding a selectable marker operably linked to a promoter thatpreferentially expresses the selectable marker in mammalian stem cells;b) culturing said mammalian cells; and c) isolating mammalian stem cellsexpressing the selectable marker.
 70. The method of claim 69, whereinthe cells obtained from the transgenic non-human animal are derived froman embryo, blood or somatic tissue.